The epidermal growth factor receptor (EGFR; HER1) is a cell surface receptor that regulates cell migration, adhesion, and proliferation. Overexpression of EGFR can be identified in many aggressive cancers such as colon, lung, and breast cancer as well as glioblastoma multiforme. EGFR is overexpressed in the cell membrane of a variety of malignant neoplasms, including colorectal, non-small cell lung carcinoma, head and neck carcinoma, and glioblastoma. EGFR has been identified as an oncogene, a gene that has the potential to cause cancer. Many targeted anticancer therapeutic approaches are aimed at the inhibition of EGFR. In order for the therapy to be effective, precise determination of the EGFR expression levels within the cell membrane is important, because only EGFR-dependent (EGFR positive) tumors respond to these therapeutic approaches.
EGFR is overexpressed in the cell membrane of a variety of malignant neoplasms, including colorectal adenocarcinoma, non-small cell lung carcinoma, head and neck carcinoma, and glioblastoma. See, Mendelsohn, J. & Baselga, J., “Epidermal Growth Factor Receptor Targeting in Cancer,” Semin Oncol 33, 369-385 (2006); Pirker, R., et al., “EGFR Expression as a Predictor of Survival for First-Line Chemotherapy Plus Cetuximab in Patients with Advanced Non-small-cell Lung Cancer: Analysis of Data from the Phase 3 FLEX study,” Lancet Oncol 13, 33-42 (2012). EGFR has been identified as an oncogene, a gene that has the potential to cause cancer. Many targeted anticancer therapeutic approaches are aimed at the inhibition of EGFR. See, e.g., Tol, J. & Punt, C. J., “Monoclonal Antibodies in the Treatment of Metastatic Colorectal Cancer: a Review,” Clin Ther 32, 437-453 (2010). EGFR inhibitor cetuximab is currently used for treating colorectal carcinoma in humans. See, e.g., Rivera, F, et al. “Cetuximab, its Clinical Use and future perspectives,” Anticancer Drugs 19, 99-113 (2008). For the therapy to be effective, precise determination of the EGFR expression levels within the cell membrane is important, because only EGFR-dependent (EGFR positive) tumors respond to these therapeutic approaches. Similarly, patients with tumors that do not express EGFR (EGFR negative) are more likely to be unaffected by the treatment. See, e.g., Vignot, S. and Spano, J., “Prognostic Value of EGFR in Colorectal Cancer. Bull Cancer 92, S13-16 (2005); Guo et al., “Overexpression of SGLT1 and EGFR in Colorectal Cancer showing a Correlation with the Prognosis,” Med Oncol 28, S197-203 (2011).
Cetuximab is an IV administered drug and costs on the order of $30,000 for eight weeks of treatment per patient. For this reason, some insurance carriers have mandated that an EGFR diagnostic test be conducted for patients before selection of an appropriate therapeutic modality.
The Dako EGFR-pharmDx™ immunohistochemistry kit has been FDA-approved as an aid in identifying colorectal cancer patients that would benefit from treatment with cetuximab and panitumumab therapies. See, e.g., U.S. Pat. No. 6,727,072. This kit is used with a multi-step process to provide a visual signal that can be analyzed with a light microscope. The kit uses immunohistochemical staining procedure with a primary monoclonal mouse antibody that selectively binds to EGFR. Primary antibodies bound to tissue antigens are detected using a peroxidase labeled polymer that is conjugated with secondary anti-mouse immunoglobulin antibodies. Enzymatic conversion of a subsequently applied chromogen forms a visible reaction product at the site of the EGFR antigen. Specimens are then counterstained and coverslipped for interpretation with a light microscope. The optical results depend upon a produced chromogen, which can provide ambiguous signals. Testing procedures must ensure control cell line staining falls within an acceptable range that is neither too light nor too dark after checking different magnifications. Weak staining intensity of a cell line can result in false-negative tests. Excessive staining can result in false positive results. The staining and analysis procedure also requires careful examination of a region or a representative area and recognition and interpretation of possible artifacts. Careful monitoring of the positive indication percentage is required to ensure that the kit and method produce valid results. The effectiveness of the kit has been examined in various publications. See, Buckley, A. F. & Kakar, S., “Comparison of the Dako EGFR pharmDx kit and Zymed EGFR Antibody for Assessment of EGFR Status in Colorectal Adenocarcinoma,” Appl Immunohistochem Mol Morphol 15, 305-309 (2007); Penault-Llorca, F., et al., “Is there an Immunohistochemical Technique Definitively Valid in Epidermal Growth. Factor Receptor Assessment?” Oncol Rep 16, 1173-1179 (2006). In practical experience, there are variations of the results obtained by different laboratories. Dako provides procedures intended to produce more consistent results. The kit's accuracy has been demonstrated to be about 51%. This could be caused by the testing procedures, or could be related to the fact that tissue fixation methodology and the quality of antibody affect results. Studies have estimated that the EGFR expression levels varied from 4% to 72% depending on the kit used. Baselga J., “Does Epidermal Growth Factor Receptor Status Predict Activity of Cetuximab in Colorectal Cancer Patients? Nature Clinical Practice Oncology 2, 284-285 (2005); Hirsch, F. R. et al., “Epidermal Growth Factor Receptor Immunohistochemistry,”. Cancer 112, 1114-1121 (2008). Another drawback is the time required for test turn-around, which is typically 24-48 hours.
Another antibody based kit was developed by Zymed (subsequently acquired by Invitrogen), and was called the EGFr Kit (clone 31G7). This test had an accuracy that was better (˜62%) than the Dako EGFR-pharmDx™ kit. However, this accuracy is still low. In addition, as another antibody based kit, the kit suffered from laboratory variation as well as sensitivity to tissue fixation methodology and quality of antibody clone. It is not believed that this kit is currently available.